Article 6 IN-VIVO ANTI-INFLAMMATORY ACTIVITY OF ... - jchps

Journal of Chemical and Pharmaceutical sciences
ISSN: 0974-2115
IN-VIVO ANTI-INFLAMMATORY ACTIVITY OF METHANOLIC EXTRACT OF
CISSUS PALLIDA
* 1 S.VIJAYA KUMAR, 2 T.SATYANARAYANA, 2 ANJANA MATHEW, 3 GANESH B, 2 VENUKUMAR.R
1 Vagdevi College of Pharmacy and Research Center, Nellore, A.P
2 University College of pharmaceutical sciences, Andhra University, Visakhapatnam, A.P
3 Sri Krishna Chaithanya College of pharmacy, Madanapalli, A.P
*Corresponding author: E mail: kumarsetty@yahoo.com
ABSTRACT
The present investigation deals with the in-vivo acute toxicity studies and in-vivo anti-inflammatory
activity by Carrageenan induced rat paw edema model of methanolic extract in the stems of Cissus pallida.
The Literature review reveals the presence of steroids, flavonoids, triterpenoids, and glycosides in Cissus
pallida. Some plants belonging to Cissus genus have been reported to posses anti-inflammatory activity
hence methanolic extract of stems of C. pallida were screened for anti-inflammatory activity. Carrageenan
induced significant inflammation when compared with the animals treated with the extracts. Standard drug,
indomethacin at 10 mg/kg inhibited the inflammation significantly at all time intervals. C.pallida exhibited
significant anti-inflammatory activity in dose dependant manner. C.pallida inhibited the carrageenan induced
inflammation significantly at doses of 500 and 1000 mg/kg at 2, 3, 4 and 5 hours.
KEY WORDS: Cissus pallida, Carrageenan induced Paw edema, Anti-Inflammatory, Acute toxicity.
1. INTRODUCTION
Plants have been used for medicinal purposes long before recorded history in many countries such as
India, China and Africa. Since then, thousands of indigenous plants have been used for the treatment of
chronic ailments. Many other herbs and minerals were later described by ancient Indian herbalists such as
Charaka and Sushruta during the 1 st millennium BC. The Sushruta Samhita attributed to Sushruta in the 6 th
century BC describes 700 medicinal plants (Agarwal,2007). In 20 th century, as part of industrial revolution,
the practice of allopathic medicines gained popularity. Eventually, the spirit of herbal medicine declined
from conventional medicinal use as safety and effectiveness of herbal medicines were not scientifically
corroborated and remain largely unknown. Due to the fact that the safety of medicine has always been
around for us, it becomes a common theme to rely on safe treatments. Usage of herbal traditional medicine is
rising steadily, because of lesser side effects, affordability and in certain diseases where no suitable
allopathic medicines are available. Based on the therapeutics significant of herbs and by proper monitoring
of quality, dosage of the drug by the ayurvedic physician is essential. Recent advances in the methodologies
used to extract, purify and evaluate plant extracts for biological activity have enable the miniaturization and
automation of extremely specific biochemical tests. As a result, in number of patients a resurgence of interest
on plants and plant derived products as a source of medicine is increasing. There is an urgent need to reemphasize
and enhance research in natural products in many therapeutic areas (Bhujbal,2008).
Inflammation term is derived from Latin word inflammare, which means - to set on fire.
Inflammation refers to a vital response of a tissue against injury elicited by physical trauma, noxious
chemical or microbiological agents. It is considered to be body’s defensive reaction either to inactivate or
destroy the injurious foreign agent or organism. It is triggered by the release of chemical mediators which
includes amines such as histamine, serotonin and lipids such as prostaglandins and small peptides such as
kinins from the injured tissues and migrating cells. Mainly, inflammatory responses occur in three distinct
phases, each apparently mediated by different mechanisms: Accordingly, pharmacological methods for antiinflammatory
evaluation have been developed by artificially inducing inflammation by using phlogistic agent
(irritants) such as: Brewer’s yeast, formaldehyde, dextran, egg albumin, kaolin, aerosol, sulfated
polysaccharides like carrageenan. The effects can be measured by several methods such as UV-erythema in
guinea pigs, Vascular permeability, Croton-oil ear edema in rats and mice, Paw edema in rats and Granuloma
pouch technique (Brooks,1991).
2. METHODOLOGY
2.1 Toxicity Studies on stems of methanolic extract of C. pallida in rats
2.1.1 Acute toxicity study in rats with test drugs: The animals were used with the approval of Institutional
Animal Ethics Committee (Reg .No. 627/02/A/CPCSEA). Two groups, each of three female rats, were
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Journal of Chemical and Pharmaceutical sciences
ISSN: 0974-2115
treated with methanolic extract of stems of C. pallida by oral administration at a dosage of 2000 mg/kg body
weight. The test drugs were formulated in vehicle (distilled water) at a concentration of 2000 mg/kg and
administered at the dose of 1ml/kg.
2.1.2 Treatment: The animals received a single dose of the test item by oral administration at 2000 mg/kg
body weight for groups I and II, after being fasted for approximately 18.0 hours but with free access to water.
Food was provided again at approximately 3.0 hours after dosing for both the groups. The administration
volume was 10 ml/kg body weight. The animals were dosed using 18 G oral Stainless steel feeding tubes.
The animals were observed daily during the acclimatization period and mortality/viability and clinical signs
were recorded. All animals were observed for clinical signs during first 30 minutes and at approximately 1,
2, 3 and 4 hours after administration on test day 0 and once daily during test days 1-14. Mortality/viability
was recorded twice daily during days 1-14 (at least once on day of sacrifice). Body weights were recorded on
test day 0 (prior to administration), test days 7 and 14. All animals were necrosed and examined
macroscopically(Dorni,2006).
2.1.3 Necroscopy: All animals were sacrificed at the end of the observation period by carbon dioxide in
euthanasia chamber and discarded after the gross/macroscopic pathological changes were visually observed
and recorded. No organs or tissues were retained.
2.2 Screening for anti-inflammatory activities of methanolic extract of stems of C. pallida in rats
2.2.1 Carrageenan induced rat paw edema model: The model is based on the principle of release of
various inflammatory mediators by carrageenan. Edema formation due to carrageenan in the rat paw is
biphasic event. The initial phase is attributed to the release of histamine and serotonin. The second phase of
edema is due to the release of prostaglandins, protease and lysosome. Subcutaneous injection of carrageenan
into the rat paw produces inflammation resulting from plasma extravasation, increased tissue water and
plasma protein exudation along with neutrophil extravasation. All these events occur as a result of
metabolism of arachidonic acid. The pharmacological screening of the C. pallida was carried out using
standard protocols 1 . The crude extract was suspended in 1% carboxy methyl cellulose (CMC) for
administration to albino rats. Albino rats of 150-200 g were used for present investigation and were used
with the approval of the Institutional Animal Ethics Committee (Regd. No. 627/02/A/CPSCSEA). They were
kept in polypropylene cages in an air-conditioned area at 25 + 2 o C in 10-14 hours light dark cycle. They were
provided with Amruth brand balanced feed and tap water ad libitum (Eshrat MH, 2003).
2.2.2 Experimental procedure: Seventy two rats were divided into five groups (n=6) starved overnight with
water adlibitum prior to the day of experiment. The group I kept as carrageenan control, groups II to IV
received test drugs at different doses and group V kept as standard drug control, respectively. Left paw was
marked with ink at the level of lateral malleolus; basal paw volume was measured plethysmographically by
volume displacement method using Plethysmometer (UGO Basile 7140) by immersing the paw till the level
of lateral malleolus. In the experiment, animals from the control group were given vehicle control, Carboxy
Methy Cellulose (CMC) and animals from standard drug were treated with Indomethacin 10 mg/kg b.w
orally as given in table 2. Other groups were treated with different doses of test drugs orally as given in table
2. After 30 min. of drug treatment the rats are charged by a subcutaneous injection of 0.1ml of 1% solution of
carrageenan into the sub-plantar side of the left hind paw (Gupta R, Bajpai KG, 2008). The paw volume is
measured again at 1, 2, 3, 4 and 5 hours after charging. The increase in paw volume is calculated as
percentage compared with the basal volume. The difference of average values between treated animals and
control group is calculated for each time interval and evaluated statistically. The percent increase in paw
volume was calculated using formula as follows.
( V )
% increase in paw volume = t
Vc
X 100
Vc
Where Vt = paw volume at a time t, Vc = paw volume at a time 0
3. RESULTS
All animals survived in group I and group II until the end of the experimental period. All the animals
dosed at 2000 mg/kg body weight did not show evident toxicity throughout the experimental period. The
animals showed increase in their body weight by day 14 as compared to day 0. No abnormalities were
detected for all the animals at necropsy. Based on the results, the median lethal doses (LD 50 ) of, C. pallida
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Journal of Chemical and Pharmaceutical sciences
ISSN: 0974-2115
was greater than 2000 mg/kg b.w and were classified as category 4 (it indicates that no death was observed at
2000mg/kg b.w.) as per OECD guidelines (Hoareau and DaSilva,1999).
4. CONCLUSION
In the control group, carrageenan induced significant inflammation when compared with the animals
treated with the extracts. Standard drug, indomethacin at 10 mg/kg inhibited the inflammation significantly at
all time intervals. C. pallida exhibited significant anti-inflammatory activity in dose dependant manner. C.
pallida inhibited the carrageenan induced inflammation significantly at doses of 500 and 1000 mg/kg at 2, 3,
4 and 5 hours.
Table.1 Body weight analysis of test drug treated rats
Test
drug
Group Dose(mg/kg
bodyweight)
Animal
Numbers
Sex Test day 0
(treatment)(g)
Testday7
(g)
Testday14
(g)
C. I 2000 002007 Female 211.83 222.95 254.06
pallida
002008 Female 206.12 231.41 261.16
002009 Female 205.04 231.78 260.85
II 2000 002010 Female 212.12 240.54 255.72
002011 Female 211.35 231.35 253.24
002012 Female 201.54 231.54 252.63
mg/kg = miligram/kilogram, g = gram
Table.2 Macroscopic findings of animals from test drug treated groups
Test
drug
C.
pallida
Group
Dose
(mg/kgbw)
I 2000
II 2000
Animal
Numbers
Sex Mode of death Macroscopic
findings
002007 Female T.S. N.A.D.
002008 Female T.S. N.A.D.
002009 Female T.S. N.A.D.
002010 Female T.S. N.A.D.
002011 Female T.S. N.A.D.
002012 Female T.S. N.A.D.
bw = body weight,T.S.= Terminal Sacrifice, N.A.D.= No abnormalities Detected
Table 3: Showing percentage of inflammation indicating Anti-inflammatory activity of test
drugs on carrageenan induced rat paw oedema
Groups Drug Doses Percentage of inflammation at time (hours)
1 2 3 4 5
I Carrageenan 38.83± 81.83± 113.3± 131.0± 146.72±
Control 1.03 1.16
2.39 4.98 5.46
II C.pallida 36.60± 79.17± 102.62± 111.52± 119.80±
250mg/kg 1.15 2.18* 6.05** 4.45*** 5.37 ***
III C.pallida 35.83± 62.57± 80.63± 93.37± 108.56±
500mg/kg 1.97** 1.74*** 3.33*** 2.84*** 3.46 ***
IV C.pallida 29.52± 57.22± 71.35± 80.55± 98.64±
1000mg/kg 1.63*** 1.62*** 1.91*** 1.42*** 2.68 ***
V
Indomethacin 12.55± 19.1± 27.17± 32.65± 30.15±
10 mg/kg 0.86*** 0.48 *** 1.07*** 1.52*** 2.50 ***
* P

ISSN: 0974-2115
Journal of Chemical and Pharmaceutical sciences
Figure.1 Percentage of inflammation indicating Anti-inflammatory activity of C. pallida in
carrageenan induced rat paw oedema
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